Non-heme, iron-containing oxygenases are involved in various biomedically important processes, including the biosynthesis of antibiotics (including chlorobiocin and penicillin), the generation of deoxyribonucleotides (key building blocks of DNA), and the storage of iron to prevent damaging Fenton-type chemistry (via the ferroxidase site of ferritin). Understanding how oxygenases use activated iron-oxygen species to carry out these transformations is of paramount importance. Despite the wealth of studies on the role of iron-oxo and iron-peroxo species, there is a paucity of spectroscopically accessible iron-superoxo compounds known. This is surprising given that iron-superoxo species are proposed as early intermediates in the catalytic cycles of almost every non-heme iron-containing oxygenase enzyme. The goal of this proposal is to generate spectroscopically tractable Fe(III)-superoxo species of relevance to non-heme iron-containing oxygenases. This will be accomplished by developing a synthetic methodology for mononuclear non-heme Fe(III)-superoxo species. We hypothesize that by appropriate combination of reaction conditions and ligand design features (including ligand basicity, steric bulk and hydrogen bond donor ability) we will be able to generate the first synthetic mononuclear non-heme Fe(III)-superoxo species. We will also apply similar design principles to the generation of Fe(III)-superoxo compounds from dinuclear precursors containing a Fe(II) center. The synthetic Fe(III)-superoxo compounds will be comprehensively characterized by a variety of spectroscopic methods including UV-vis, XAS, resonance Raman, EPR, and Mssbauer spectroscopy. This will allow the establishment of an understanding of the relationship between primary- and secondary- coordination sphere features of the compounds to the structural and electronic features of iron-superoxo species. To date no non-heme iron-superoxo compound has been fully characterized by a combination of Raman, EPR and Mssbauer spectroscopy, and this is a significant gap in understanding for the field. In fact, only a single such superoxo compound has been characterized by resonance Raman, and the ?O-O stretching frequency of 1310 cm-1 is anomalously high compared to other known superoxide compounds (1043-1207 cm-1). The reactivity of the synthesized Fe(III)-superoxo compounds will also be explored in order to establish a reactivity relationship to electronic and structural parameters. Overall, this research has the potential to be transformative to understanding the role of superoxide intermediates in the field of non-heme iron-containing oxygenases.